基于双边拟合的高稳定性共焦拉曼光谱定焦方法

共焦拉曼光谱技术可实现定量、无损、无需标记的样品微区“分子结构特征和物质组成信息”成像，被广泛应用于生物医学、物理化学以及材料科学等领域。由于共焦拉曼系统采用“点”激发和“点”探测的探测机制，且拉曼散射光谱信号微弱，导致成像所需时间可长达数小时甚至数十小时；测量过程中系统极易受环境变化、空气扰动等因素影响产生漂移，造成被测样品离焦，从而导致成像质量不稳定。针对现有共焦拉曼系统对样品定焦能力不足、样品易产生离焦误差、系统漂移大等问题，本文提出了一种基于双边拟合的高稳定性共焦拉曼光谱定焦方法。该方法首先对共焦拉曼光谱强度轴向响应曲线两侧对样品离焦敏感的数据区间分别进行线性拟合，得到两条拟合直线方程；然后，将所得的两条直线方程相减得到新的差分直线；最后，通过差分直线的过零点位置确定系统焦平面位置，实现了被测样品的高精度定焦，消除了离焦对系统测量结果的影响。以单晶硅表面同一位置，轴向扫描步距100 nm，进行60次重复定焦实验，实验获得的重复定焦极差为80.2 nm，说明系统具有良好的抗漂移能力。对周期5 μm的竖条栅格标准原子力台阶样品进行拉曼mapping成像测试，结果表明在长时间的成像过程中，和无定焦功能的图像相比，该方法获得的竖条栅格图像更清晰、边缘更锐利、信噪比较好。仿真分析和实验结果表明：提出的基于双边拟合共焦拉曼光谱探测方法可以提高系统的定焦准确度，抑制干扰因素导致的系统离焦对成像质量的影响，进而确保了系统探测的稳定性和成像分辨力，是一种自动定焦、抗漂移的拉曼光谱成像方法。

High-Stability Confocal Raman Microscopy Imaging Method Based on Bilateral Fitting Subtracting Confocal Microscopy

Confocal Raman microscopy can realise quantitative, nondestructive, and unlabeled imaging for sample microregions and obtain molecular structure characteristics. The time consumption of the confocal Raman microscopy system always lasts up to several hours or even tens of hours. During the time-consuming measurement process, it is highly vulnerable to some disturbing factors. These factors lead to the system’s shift and make the sample out-of-focus, which results in poor imaging quality. This paper proposes a high-stability confocal Raman microscopy based on bilateral fitting subtraction to solve the above issues of the existing confocal Raman microscopy. Firstly, the proposed method utilizes the linear fittings about the data areas on both sides where the axial response of the confocal Raman spectral intensity curve is sensitive to the defocus of the sample position. The expressions of the two equations about the linear fittings are obtained. Secondly, through subtracting these two expressions, a differential expression can be obtained. The zero-crossing of the differential expression is the focal plane position of the confocal Raman microscopy. Monocrystalline silicon is used to test the focusing repetition accuracy of the proposed method. The monocrystalline silicon surface is axially scanned with a scanning pitch of 100 nm and the experiment is repeated 60 times. The results show that the maximum difference is 80.2 nm, which indicates that the proposed system exhibits a strong anti-drift ability. We also performed a Raman mapping imaging test on a standard atomic force step sample with a bar lattice of a period of 5 m. Compared with the image obtained by the confocal Raman microscopy without auto-focusing ability, the image obtained by the proposed confocal Raman microscopy is clearer, and the edge of the image is sharper as well, as a high signal-noise ratio. The simulation analysis and experimental results show that the proposed confocal Raman microscopy based on the bilateral fitting subtraction method can improve the focus ability and suppress the defocus effect caused by interference factors on the image quality. The proposed method provides a guarantee for obtaining a confocal Raman spectrum image with high stability and offers a new avenue of Raman spectral imaging method for automatic focusing and anti-drift.